BMB
Reports
Invited Mini Review
Body fluid identification in forensics
Ja Hyun An , Kyoung-Jin Shin , Woo Ick Yang & Hwan Young Lee *
1 1,2 1 1,2,
1
Department of Forensic Medicine, Yonsei University College of Medicine, 2Human Identification Research Center, Yonsei University,
Seoul 120-752, Korea
Determination of the type and origin of the body fluids found
at a crime scene can give important insights into crime scene
reconstruction by supporting a link between sample donors
and actual criminal acts. For more than a century, numerous
types of body fluid identification methods have been
developed, such as chemical tests, immunological tests,
protein catalytic activity tests, spectroscopic methods and
microscopy. However, these conventional body fluid
identification methods are mostly presumptive, and are carried
out for only one body fluid at a time. Therefore, the use of a
molecular genetics-based approach using RNA profiling or
DNA methylation detection has been recently proposed to
supplant conventional body fluid identification methods.
Several RNA markers and tDMRs (tissue-specific differentially
methylated regions) which are specific to forensically relevant
body fluids have been identified, and their specificities and
sensitivities have been tested using various samples. In this
review, we provide an overview of the present knowledge and
the most recent developments in forensic body fluid
identification and discuss its possible practical application to
forensic casework. [BMB Reports 2012; 45(10): 545-553]
INTRODUCTION
Biological samples found at the crime scenes play pivotal roles
in forensic investigations by providing valuable evidence (1).
Since the DNA profile of every individual is considered to be
unique (except in the case of identical twins), DNA typing of
biological samples can prove whether a suspect was involved
in a crime, and even can exonerate innocent persons who
have been wrongfully convicted (2). Besides, determination of
the type and origin of biological samples found at crime
scenes can provide important clues for crime scene re-
constructions by supporting a link between sample donors and
*Corresponding author. Tel.: +82-2-2228-2482; Fax: +82-2-362-
0860; E-mail: hylee192@yuhs.ac
http://dx.doi.org/10.5483/BMBRep.2012.45.10.206
Received 20 September 2012
Keywords: Alternate light source, Body fluid identification,
Catalytic test, Chemical test, DNA methylation, Forensic, Immuno-
logical test, miRNA, mRNA,
ISSN: 1976-6696 (print edition) / 1976-670X (electronic edition)
Copyright 2012 by the The Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/li-
censes/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
actual criminal acts. The presence of certain body fluids can
be used as excellent indicators of the sequence of events
which occurred. For example, blood stains can indicate some
form of physical struggle, assault or murder, and detection of
semen or vaginal fluid can indicate the involvement of some
form of sexual encounter or assault. The common body fluids
found at crime scenes are blood, semen, saliva, vaginal fluid,
urine, and sweat.
When a potential body fluid is discovered at a crime scene,
a particular form of light or chemical addition may be required
to visualize the stain (3). Presumptive tests are initially used to
give some indication as to the identity of the substance, and
further confirmatory tests are then conducted to confirm the
origin of the sample (2). For more than a century, numerous
types of analysis methods have been developed for inves-
tigation of forensically relevant body fluids. Techniques which
are currently used for forensic body fluid identification include
chemical tests, immunological tests, protein catalytic activity
tests, spectroscopic methods, and microscopy. Some of these
methods are presumptive tests which are used as screening
tests, such as the luminol and Kastle-Meyer test for blood.
Others are confirmatory test that will conclusively identify the
presence of certain body fluid, such as the microscopic identi-
fication of sperm cells (4, 5). Most catalytic, enzymatic and im-
munologic tests which have been used for presumptive or con-
firmatory methods suffer from several limitations, such as low
specificity, lack of sensitivity, sample destruction, instability of
biomolecule assayed, or incompatibility with downstream in-
dividual identification assays although they have certain ad-
vantages (2).
Recent advances in forensic genetics have led to the devel-
opment of several new methods, and the majority of these
methods involve the detection of specific messenger RNA
(mRNA) and micro RNA (miRNA) expressions, as well as dif-
ferential DNA methylation patterns. Especially, recent ap-
proaches based on tissue-specific mRNA or miRNA expression
have been proved to be useful because of their high tissue spe-
cificity to forensically relevant body fluids. However, ubiq-
uitously present ribonucleases are detrimental to mRNA stabil-
ity, and RNA typing requires additional sample material, un-
less the RNA is co-extracted with DNA. More recently,
DNA-methylation based assays, which identify differential
DNA methylation profiles of different cell or tissue types, have
been proposed as a promising new method for distinguishing
 Body fluid identification in forensics
Ja Hyun An, et al.
between different types of body fluids because of their high
specificity and fit with current forensic casework application.
This review briefly describes current and previous techni-
ques of body fluid identification that are being used in forensic
laboratories, and evaluates the advantages and disadvantages
of each method. We will also focus on new methods of identi-
fication that have been developed in the past decade, specifi-
cally mRNA based assays, miRNA based assays and DNA
methylation based assays. The review concludes with a dis-
cussion of the potential application of these new methods for
forensic case works.
CURRENT TECHNIQUES
Alternate light source methods
The simplest way to detect body fluid stains that are difficult to
see with the naked eye is to use an alternate light source (ALS)
such as ultraviolet light. Because it is a routine procedure to
search a crime scene for latent stains of body fluids using ALS,
there are various commercial ALS devices, such as Woods
Lamp (WL), specific for semen detection. WL which emits
wavelengths ranging about 320-400 nm is safe and handy, but
the specificity is somewhat low, thereby carrying a high risk of
false positive results for other fluids (6). Another ALS device,
TM
Bluemaxx BM 500 demonstrated 100% sensitivity to semen
stains (7), and Polilight can detect several body fluids, includ-
ing semen (8). A newly developed device, Lumatec Superlight
400 emits light from 320 to 700 nm and is able to detect stains
both in darkness and in daylight (9). Using this device, semen
was best detected using a range of 415-490 nm, and saliva was
also detectable in 60% of cases. However, poor results were
obtained when it was applied to dark fabrics and to fabrics
which had been washed, although it had been reported that dif-
ferent types of fabrics showed similar results (9, 10).
Chemical tests
Chemical tests are usually based on the color change or chem-
iluminescence of a particular reagent when it comes into con-
tact with a particular body fluid. Luminol is frequently used in
identifying blood stains, particularly when the perpetrator has
attempted to clean up the blood, thus rendering it invisible to
the naked eye (11, 12). Due to the emission of light as a result
of an oxidation of luminol enhanced by iron in hemoglobin
and its derivatives in blood, trace amounts of blood can be de-
tected by chemiluminescence of a blue-green color (13, 14).
However luminol has been known to react with other sub-
stances, including copper containing chemical compounds,
certain bleaches, saliva, and various animal and vegetable
proteins. The luminol test remains popular due to the rela-
tively high sensitivity and specificity in comparison with other
screening tests. However, it is limited to use in dark environ-
ments and also has disadvantages regarding the duration of il-
lumination (about 30 seconds) and sometimes has detrimental
effects on subsequent DNA analysis (15-17). Bluestar Foren-
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sic is another latent blood stain reagent based on chemilu-
minescence, and it gives more sensitive and stable results
without damaging DNA and thereby allows for subsequent
genotyping of stains (16, 18).
Catalytic tests
Catalytic methods are based on the enzyme activity which cat-
alyzes the reaction of a variety of substrates to produce visible
color changes. There are several different catalytic tests which
are commonly used to presumptively identify blood based on
the peroxidase-like activity of heme group (13, 14). The heme
group of hemoglobin possesses a peroxidase-like activity
which catalyzes the breakdown of hydrogen peroxide, and the
oxidizing species formed in this reaction can then react with a
variety of substrates to produce a color change (19). Among
the substrates in common use are benzidine and various sub-
stituted benzidines, ortho-tolidine, leucomalachite green, leu-
cocrystal violet and phenolphthalein, also known as the
Kastle-Meyer test (20, 21).
The most commonly utilized of these tests is the benzidine
test, where the presence of blood is indicated by blue colored
products. However, there are several substances which can
generate false positives for this test, such as chemical oxidants
and fruit and vegetable peroxidases (22). Moreover, benzidine
is known as a carcinogen (23), and accordingly this has largely
been replaced by tests using phenolphthalein/hydrogen
peroxide. The test using phenolphthalein is also known as the
Kastle-Meyer test, which is a very popular presumptive test for
blood. The stain in question is collected with a swab or filter
paper, and phenolphthalin reagent and hydrogen peroxide are
then applied to the sample. If the sample contains hemoglo-
bin, a result produces a pink coloring only after the addition of
hydrogen peroxide, by oxidation of phenolphthalin into
phenolphthalein. Although false-positives are reported in the
presence of chemical oxidants and vegetable peroxidases, the
test can detect blood as dilute as 1 part in 10,000. In addition,
this test is nondestructive to the sample, which can then be
kept and used in further tests, including DNA analysis (20, 21,
24, 25).
The acid phosphatase (AP) test is one of the most common
tests for semen detection. AP is an enzyme secreted by the
prostate gland that is present in large amounts in seminal fluid
(26). The level of AP activity is 500 to 1,000 times higher in
human semen than in any other body fluid. AP can catalyze
the hydrolysis of phosphates, which results in the formation of
a product that will react with the color developer (27).
Therefore, in the presence of Alpha-Naphthyl acid phosphate
and Brentamine Fast Blue, AP will produce a dark purple color
(27). However, the test for AP is highly presumptive because
vaginal secretions and other body fluids also contain detect-
able levels of this enzyme (13).
A catalytic test for the detection of saliva is based on the en-
zymatic activity of alpha-amylase. Saliva is rich in alpha-amy-
lase, an enzyme that hydrolyses polysaccharides into smaller
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sugar molecules (28). The starch-iodine detects the alpha-amy-
lase activity owing to the fact that iodine reacts with the starch
producing a purple black color. In the presence of the amy-
lase, the intensity of color decreases as starch is broken down
by any amylase present (2). Another method for the detection
of alpha-amylase is the Phadebas test, which includes amylo-
pectin-procion red instead of starch-iodine (29, 30). This test is
relatively cheap, quick, and highly sensitive, but false positive
results were also observed in hand cream, face lotion, urine,
and feces (29, 30). Additionally, there are two different forms
of alpha-amylase in the human body; AMY1 found in saliva,
breast milk and perspiration and AMY2 found in the pancreas,
semen and vaginal secretions. Although AMY1 is found pre-
dominantly in saliva compared with other types of body fluids,
the two variants are almost indistinguishable in terms of their
enzyme activity. Thus, detection of the activity of alpha-amy-
lase can only give presumptive information because it is not
exclusive to saliva (2, 27).
Immunological tests
Many traditional tests used in forensic science are immuno-
logical tests that are based on specific antibody-antigen
reaction.
The OneStep ABAcard HemaTrace test strip is an im-
munochromatographic test for the detection of human blood
(2, 14, 25). If human hemoglobin is present in the sample, it
will combine with a mobile monoclonal anti-human hemoglo-
bin antibody in the test strip. Any antibody-antigen complex
formed then migrates through an absorbent membrane to the
test area. When the human hemoglobin concentration is
above a certain minimum detection limit (0.05 g/ml), the
pink dye becomes visible in the test region. The results have
shown the OneStep ABAcard HemaTrace to be a highly sen-
sitive, convenient, and rapid test for the identification of hu-
man blood both in the laboratory and at crime scenes.
The most commonly used confirmatory test for semen de-
tection beyond microscopic identification of the sperm cells is
a test based on the detection of prostate specific antigen (PSA)
(31). PSA is produced in high amounts by the male prostate
gland, and is even detected in the semen from azoospermic
males (27, 32). Commercial test kits that depend on anti-
body-antigen reaction are currently widely used for PSA
detection. One of the most popular commercial kits, the
OneStep ABAcard PSA, also uses the technique of a mobile
monoclonal anti-human PSA antibody which binds to human
PSA and migrates along the strip, forming a visible line in its
path (33, 34). However, studies have shown that PSA can also
be found in very low levels in other body fluids, such as fe-
male urine and breast milk (35, 36).
EMERGING TECHNIQUES
Messenger RNA profiling methods
During the last decade, RNA analysis has emerged as a prom-
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Body fluid identification in forensics
Ja Hyun An, et al.
ising new tool for forensic investigation applications, such as
inferences about post-mortem intervals and wound age (37).
Additionally, recent development in forensic genetics revealed
that tissue-specific RNA expression can be used for human
body fluid identification.
RNA is notorious for its instability because of the ubiq-
uitously present ribonucleases. However, recent studies have
reported that RNA isolated from some forensic stains showed
unexpectedly high stability. Using whole-genome gene ex-
pression on aged blood and saliva stains, Zubakov et al. (38)
identified blood and saliva specific messenger RNA (mRNA)
markers that showed stable expression patterns in stains after
up to 180 days of storage, and some of these markers showed
successful and reliable amplification in much older stains,
such as 16 year-old blood stains (39). Setzer et al. (40) con-
ducted a more comprehensive study on mRNA stability in for-
ensic samples. They exposed biological stains to a range of en-
vironmental conditions and performed mRNA profiling analy-
sis using eight different mRNA transcripts of selected house-
keeping and tissue-specific genes. The results demonstrated
that RNA is detectable in some samples stored at room temper-
ature, even after 547 days, but heat and humidity appear to be
detrimental to RNA stability.
One of major advantages of body fluid identification by
mRNA profiling is the possibility of simultaneous extraction of
mRNA and DNA from the same stain, posing a major advant-
age in forensic investigations, where sample material is often
limited. Actually, several optimized methods have been devel-
oped for simultaneously isolating mRNA and DNA from same
physical stains (41, 42), and successful identification of the
stain could be performed from the same stain where the do-
nors identity was confirmed (43, 44). Another important ad-
vantage of mRNA profiling is the possibility of detecting sev-
eral body fluids in one multiplex reaction, providing data on
the expression of multiple genes simultaneously.
Various multiplexes have been reported, using reverse tran-
scription endpoint polymerase chain reaction (RT-PCR) meth-
ods and real-time quantitative reverse transcriptase-PCR
(qRT-PCR) assay, including body fluid-specific markers (43-48).
Since many transcripts are not completely tissue specific but
usually show differences in expression level, qRT-PCR might
be more appropriate for the detection of relative gene ex-
pression levels in different samples, and endpoint PCR might
be suitable for the detection of certain transcripts with highly
tissue-specific expression (48, 49).
In 2005, Juusola and Ballantyne (45) proposed a multiplex
RT-PCR method for the identification of body fluids that are
commonly encountered in forensic casework. Using eight se-
lected body fluid-specific genes, i.e., -spectrin (SPTB) and
porphobilinogen deaminase (PBGD) for blood, statherin
(STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1)
and protamine 2 (PRM2) for semen, and human beta-defensin
1 (HBD-1) and mucin 4 (MUC4) for vaginal secretions, they
were able to detect each of the four body fluids as single or
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Ja Hyun An, et al.

mixed stains. The sensitivity of the system was suitable for for-
ensic casework because successful identification of body fluids
was possible with 200 pg-12 ng of input RNA. In the updated
experiment in 2007, Juusola and Ballantyne (46) reported mul-
tiplex qRT-PCR assays for the identification of blood, saliva, se-
men, and menstrual blood. In this work, previously identified
body fluid-specific genes were used for saliva and semen, but
blood specific marker PBGD was replaced with -amino-
levulinate synthase 2 (ALAS2). In addition, matrix metal-
loproteinase 7 (MMP7) and matrix metalloproteinase 10
(MMP10) were added for the detection of menstrual blood,
and housekeeping gene GAPDH was introduced for the nor-
malization of the expression of body fluid-specific genes.
Nussbaumer et al. (47) also reported multiplex qRT-PCR as-
says, which include hemoglobin A (HBA) for blood, kallikrein
(KLK) for semen and mucin 4 (MUC4) for vaginal fluid or
saliva. In the results, all semen samples showed a high ex-
pression of KLK mRNA, while none of other body fluids
showed any expression of KLK. On the other hand, HBA
mRNA was highly expressed in all blood samples screened,
but some of the saliva samples also showed a weak reaction,
while still being discriminative in expression level. Haas et al.
(48) reported a multiplex RT-PCR and a multiplex qRT-PCR for
the identification of blood, saliva, semen, menstrual blood and
vaginal fluid using previously reported mRNA markers; SPTB,
PBGD, STATH, HTN3, PRM1, PRM2, HBD1, MUC4, MMP7
and MMP11 (Fig. 1). The two developed multiplex RT-PCRs
showed high specificity, sensitivity and suitability for forensic
body fluid analysis, and mRNA stability was demonstrated for
up to 2-year-old stains. Recently, a collaborative exercise on
mRNA profiling for the identification of blood was organized
by the European DNA Profiling Group (EDNAP) (43), and the
results demonstrated that all but one of the 16 participating
laboratories were able to successfully isolate and detect blood
specific-mRNA from the dried blood stains.
Incidentally, several mRNA markers proposed for the identi-
fication of vaginal secretions show controversial results leading
to false positives in saliva samples (45, 47, 50). Hence,
Fleming and Harbison (51) suggested improving the mRNA
multiplex system for body fluid identification by applying mi-
crobial markers for vaginal fluids. They have incorporated
16S-23S rRNA intergenic spacer region (ISR) of Lactobacillus
crispatus and Lactobacillus gasseri, reportedly predominant in
the vagina of women, into previously described mRNA
multiplex. From their results, the 16S-23S ISR of L. crispatus
and L. gasseri were detected in vaginal secretions and in some
menstrual blood samples but not in any other fluids, including
blood, semen, and saliva. Although further works are needed Fig. 1. (A) Endpoint PCR multiplex 1 result of a blood-saliva-se-
for samples of various age ranges, this study provided the po- men-vaginal secretion-menstrual blood mixture, revealing two
peaks for each body fluid. Electropherograms of blood (B), vaginal
tential use of vaginal bacteria as a tool for the identification of secretion (C) and menstrual blood (D) samples detected with end-
vaginal fluids. point PCR multiplex 2. Reprinted from Haas et al. (48) with kind
permission from Elsevier.
MicroRNA profiling methods
Although a number of mRNA markers have been proposed as

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sensitive and specific methods for forensic body fluid determi-
nation, and have also proven their stability successfully in sam-
ples over long periods of time, additional environmental con-
ditions such as humidity and temperature are expected to in-
fluence mRNA stability (39, 40). Therefore, recently, micro-
RNA (miRNA) markers have started to be explored as an alter-
native tool for forensic body fluid identification (52-54).
MiRNAs are non-coding RNA molecules of 18 to 22 nucleo-
tides in length, and regulate gene expression at the post-tran-
scriptional level (55-57). Several recent studies have demon-
strated that many miRNAs showed tissue-specific expression
patterns (58, 59). In addition, the intrinsically small size of
miRNAs makes them less prone to degradation by environ-
mental factors, thus offering an obvious advantage as a useful
biomarker for body fluid identification (52). In 2009, Hanson
et al. (52) provided the first introduction of miRNA profiling to
forensic science, and evaluated the miRNA expression in for-
ensically relevant biological fluids. In the study, they demon-
strated that miRNA can be extracted from forensic samples and
examined the expression of 452 human miRNAs in blood, sali-
va, semen, vaginal secretions and menstrual blood using
qRT-PCR analysis (miScript SYBR Green PCR kit, Qiagen).
They have identified nine miRNAs-miR451, miR16, miR135b,
miR10b, miR658, miR205, miR124a, miR372, and miR412-
that are differentially expressed in forensic biological samples,
and have proven their abilities to identify body fluid using as
little as 50 pg of total RNA. Zubakov et al. (53) also profiled
the expression levels of 718 miRNAs in forensically relevant
body fluids using genome-wide microarrays and identified 14
differentially expressed candidates for potential body-fluid
identification. In the subsequent validation test using qRT-PCR,
only blood and semen specific miRNA candidates showed
comparable expression levels in microarray and qRT-PCR data,
whereas less concordance was found for saliva, menstrual
blood and vaginal secretions. Although these two initial stud-
ies showed the potential of miRNA profiling for forensic inves-
tigation, their results were inconsistent when different technol-
ogy platforms and statistical methods were applied. Therefore,
it has been suggested that rigorous methodological validation
and accurate models for data analysis are needed for accurate
miRNA quantification in forensic applications (54).
DNA methylation profiling methods
Since recent whole-epigenome analyses indicated that DNA
carries tissue-specific methylation patterns, the potential of tis-
sue-specific differential DNA methylation for body fluid identi-
fication has been examined in the forensic field (60-65). DNA
methylation, which occurs at the 5'-position of the pyrimidine
ring of cytosine in CpG dinucleotides, is generally believed to
inhibit gene expression by affecting chromatin structure (66,
67). Different cell types have different methylation patterns
(68), and chromosome segments called tissue-specific differ-
entially methylated regions (tDMRs) are known to show differ-
ent DNA methylation profiles according to cell or tissue type
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Body fluid identification in forensics
Ja Hyun An, et al.
(69, 70). Thus, detection of DNA methylation status at a cer-
tain CpG site of tDMR would allow for identification of the tis-
sue or cell type of DNA samples.
In 2011, Frumkin et al. (60) reported 15 genomic loci which
are differentially methylated among blood (venous/menstrual),
saliva, semen, skin epidermis, urine and vaginal secretion. In
this study, they implemented an assay for selected markers us-
ing methylation-sensitive restriction enzyme-PCR (MSRE-PCR)
made up of methylation-sensitive restriction enzyme digestion
of sample DNA followed by multiplex PCR of specific ge-
nomic loci with fluorescence-labeled primers, capillary elec-
trophoresis of amplification products and automatic signal
detection. The assay can be easily integrated into standardized
procedures of forensic laboratories like DNA typing using
short tandem repeat (STR), and could successfully identify
source tissues in 50 DNA samples from blood, saliva, semen,
and skin epidermis. Later, Wasserstrom et al. (63) advanced
the previously reported approach by developing a kit, DNA
TM
source identifier (DSI)-Semen , which aims to replace micro-
scopic examination of sperm cells for forensic semen identi-
fication in casework samples. This assay is based on the de-
tection of semen-specific DNA methylation patterns in five ge-
nomic loci using MSRE-PCR. The kit was validated with 135
samples of various body fluids and 33 actual casework sam-
ples from the forensic biological laboratory, and was proven to
be robust and reliable by showing a positive result for semen
given as little as 31 pg of template DNA input (63, 64).
Lee et al. (61) also examined the potential of tDMRs for for-
ensic body fluid identification using a bisulfite sequencing
method. Bisulfite sequencing determines the DNA methylation
status by detection of nucleotide base change due to sodium
bisulfite treatment. Bisulfite treatment has no influence on me-
thylated cytosine, but converts free or unmethylated cytosine
of CpG to uracil, which becomes thymine during subsequent
PCR. Using this method, they produced methylation profiles
for five tDMRs in pooled DNA samples from blood, saliva, se-
men, menstrual blood, and vaginal fluid. The tDMRs for
DACT1 and USP49 were selected as a semen-specific marker
by showing semen-specific hypomethylation, and the PFN3
tDMR was suggested to be used for vaginal fluid identification.
In a recent paper, An et al. (62) further investigated age-related
methylation changes in semen-specific tDMRs using body flu-
ids from young and elderly man, since DNA methylation pat-
terns are known to be susceptible to change by aging (71,72).
After confirming the stability of the body fluid-specific DNA
methylation profile, they proposed two multiplex systems to
analyze the methylation status of the USP49, DACT1, PRMT2
and PFN3 tDMRs. The two multiplex systems were con-
structed using MSRE-PCR and methylation SNaPshot, and both
could successfully identify semen with sperm cells and could
distinguish menstrual blood and vaginal fluids from other body
fluids in a test with 144 DNA samples (Fig. 2). Unlike MSRE-
PCR, which shows only the amount of methylated CpGs,
methylation SNaPshot has a merit that can measure the pro-
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 Body fluid identification in forensics
Ja Hyun An, et al.
portion of the methylated and/or unmethylated cytosine of the
target CpG site simultaneously, because this assay is carried
out by the amplification of bisulfite-converted DNA and sub-
sequent single base extension reaction. On the other hand,
since genomic DNA can be degraded during bisulfite treat-
ment, a bisulfite-based methylation SNaPshot assay may con-
sume more samples than MSRE-PCR. Therefore, a sensitivity
test was performed for the multiplex methylation SNaPshot,
and the result showed that a minimum of 500 pg of starting ge-
nomic DNA, or 125 pg of bisulfite-converted DNA, was suffi-
cient for successful DNA methylation profiling of the selected
tDMRs, which demonstrates the possible practical application
of the multiplex system to forensic casework.
A recent paper by Madi et al. (65) also reported tissue-spe-
cific DNA methylation in forensically relevant biological sam-
ples including blood, saliva, semen and epithelial cells. They
examined a few genomic loci using bisulfite modification and
pyrosequencing to find that the methylation patterns at the
ZC3H12D and FGF7 loci can differentiate sperm from other
biological samples while the C20orf117 locus and the BCAS4
locus can differentiate blood and saliva from other samples,
respectively. These results also indicate that the DNA methyl-
ation-based methods could be a valuable analysis tools for the
characterization of forensically relevant biological fluids, but
further validation studies including more markers will be re-
quired for actual casework applications.
CONCLUDING REMARKS
While current forensic DNA profiling based on STRs allows
personal identification of biological sample donors, recent ad-
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Fig. 2. Typical DNA methylation pro-
files for samples from blood (A), sali-
va (B), menstrual blood (C), vaginal
fluids (D) and semen (E) using MSRE-
PCR (left) and methylation SNaPshot
(right). Reprinted from An et al. (62)
with kind permission from Springer
Science + Business media.
vances in forensics has suggested using other types of markers
in order to add more informative layers to the evidence.
Especially, various kinds of markers have been proposed for
forensic body fluid identification. Among emerging techniques
we have discussed, mRNA markers have been most rigorously
investigated and the number of specific markers is sufficient
for the identification of forensically relevant body fluids. Some
of the other currently used methods have potential for the rap-
id and non-destructive identification of body fluids, but in
most cases, tissue specificity is problematic due to cross re-
action with biological or non-biological material present in
samples at crimes scene. On the other hand, mRNA markers
show high tissue-specificity and adequate sensitivity for foren-
sic analysis, thereby being considered as a valuable new ap-
proach to overcome the limitations of conventional methods.
However, mRNA profiling is not yet widely used in forensic
laboratories probably owing to the fact that it does not allow
for the simultaneous analysis of DNA profiling and body fluid
identification. In addition, heat and humidity remain threats to
the stability of mRNA markers. Recently, DNA methylation
profiling was proposed as a promising new tool for forensic
body fluid identification, which uses the same DNA samples
which are used for DNA typing. Like mRNA profiling meth-
ods, DNA methylation profiling showed high specificity and
sensitivity, and also allowed for the simultaneous analysis of
multiple markers specific for various tissues in a single multi-
plex system. Moreover, DNA methylation profiling methods fit
well with current forensic applications, and accordingly, can
be easily integrated into forensic standardized procedures.
However, excepting semen applications, DNA methylation
still needs to identify more markers for future practical applica-
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tion to casework. We believe that it is now possible to identify
more informative markers for body fluid identification using
various high-throughput screening technologies. In the near fu-
ture, forensic investigations should improve a great deal be-
cause of the continued advances in genetics, epigenetics and
molecular biology, and hence, the extraction of more in-
formation from forensically relevant biological samples will be
possible.
Acknowledgements
This study was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (2010-0005208) and a grant from the National
Forensic Service (NFS) funded by the Ministry of Public
Administration and Security.
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